A2AR基因敲除对小鼠脑缺血再灌注后SVZ区星形胶质细胞激活的影响

Effect of A2AR Knock out on Astrocyte Activation in SVZ of Focal Cerebral Ischemia-reperfusion in Mice

通过研究腺苷A2A受体(A2A receptor,A2AR)基因敲除对小鼠局灶性脑缺血再灌注后侧脑室下区(subventricular zone,SVZ)星形胶质细胞(astrocyte,Ast)数目及形态的影响,为腺苷类药物在脑卒中类疾病中的应用提供实验依据。采用大脑中动脉栓塞法(middle cerebral artery occlu-sion,MCAO)复制局灶性脑缺血再灌注模型,动物分为假手术组(S)和模型组(M),其中M组根据基因类型分为A2AR基因敲除模型组(MKO)和A2AR基因野生模型组(MWT),MKO组和MWT组根据再灌注时间都分为1 d组、3 d组和7 d组,S组分为A2AR基因敲除假手术组(SKO)和A2AR基因野生假手术组(SWT),共8组;对实验小鼠进行神经行为学评分,脑组织用2,3,5-氯化三苯基四氮唑(2,3,5-triph-enyltetrazolium chloride,TTC)染色观察脑梗死灶,苏木素伊红(HE)染色和尼氏(Nissl)染色观察脑组织大体形态,以及应用免疫荧光法观察SVZ区Ast的特异性标记物胶质纤维酸性蛋白(glial fibrillaryacidic protein,GFAP)表达。结果显示,所有M组小鼠在脑缺血再灌注后均出现神经功能缺损症状,神经行为学评分显示MKO 3 d组低于MKO 1 d组(P<0.05);TTC显示M组小鼠脑组织均有白色梗死灶;免疫荧光结果显示S组小鼠SVZ区域仅有少量Ast表达,M组表达明显增多,MKO 7 d组Ast表达量低于MWT 7 d组(P<0.05);Ast表达与神经行为学评分呈显著负相关(r=–0.621,P<0.01)。结果证明,A2AR基因敲除对小鼠局灶性脑缺血再灌注后,在急性期能够减轻肢体活动障碍,在恢复期能够调控Ast的激活,减少胶质瘢痕形成,是其促进后期神经再生的可能因素之一。

This article investigates the effects of adenosine A2A receptor （A2AR） on astrocyte （Ast） acti- vation in subventricular zone （SVZ） during focal cerebral ischemia-reperfusion in mice, to provide experimental evidences for application of adenosine drugs on Stroke. Focal cerebral ischemia-reperfusion was induced by middlecerebral artery occlusion （MCAO）. The mice were randomly divided into Sham operated groups （S）, model groups （M）, and the model groups were divided into A2AR-gene-ko-model-groups （MKO） and A2AR-gene-wt-model- groups （MWT）. According to the time after ischemia-reperfusion, MKO groups and MWT groups were divided into 1 d groups, 3 d groups and 7 d groups. And the S groups were divided into A2AR-gene-ko-sham-groups （SKO） and A2AR-gene-wt-sham-groups （SWT）. Neurological behavior was assessed on each mice. Brain slices were observed for infarction by 2,3,5-triphenyltetrazolium chloride （TTC）; general shape of brain was observed by HE＆Nissl staining; and the specific markers glial fibrillary acidic protein （GFAP） of Ast was measured by Immunofluores- cence. Abnormal neurological behavior was observed in the animals of M groups, but the neurological behavior of mice in MKO 3 d group was better than these in the MKO 1 d group （/＇〈0.05）. Typical cortical infarct lesion in M groups was found by TTC staining. The expression of GFAP in the brain of S groups was rather little, which was enhanced significantly in M groups; besides, the expression of GFAP in the MKO 7 d group was less than that in the MWT 7 d group. A2AR-gene-ko can mitigate handicap during acute stage of ischemia reperfusion, regulate ac- tivation of astrocyte during convalescence, which should be one of favorable factors for neuranagenesis.